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Within the state of Florida, there are three arboviruses of public health importance that can cause neuroinvasive disease in humans: West Nile Virus, Saint Louis Encephalitis Virus, and Eastern Equine Encephalitis Virus. Mosquitoes (Diptera: Culicidae) within the genus Culex are known and suspected vectors of these diseases. The vectors of these diseases can be present in urban wetland habitats that allow for exposure to residential communities. Vector ecology must be investigated in order to understand the dynamics of disease transmission. In Hillsborough County, Florida the spatial and temporal distribution of these vectors are not well established. An ecological study was conducted in the University of South Florida's Ecopreserve using trapping methodologies to sample the adult and gravid females as well as collect the egg population. Collections were made at three spatial points for the duration of July through December 2013 and compared to meteorological variables. Culex erraticus, a proposed bridge vector of Eastern Equine Encephalitis, was the most abundant adult species and gravid female captured. Culex nigripalpus, primary Floridian vector of Saint Louis Encephalitis and bridge vector of West Nile Virus, was the second most abundant adult species caught as well as the majority of eggs collected. Based on the results collected, the presence of Culex erraticus and Culex nigripalpus was confirmed. The majority of Culex erraticus adults were collected in September and October and Culex nigripalpus adults were the highest in July and August. The results of the gravid and egg collection generated crucial insight regarding methodology for studying vector ecology within this urban wetland habitat. However, modeling at spatial points based on meteorological variables yielded inconsistent results that illicit further investigation regarding these arboviral vectors of disease.

West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging mosquito-borne flaviviruses. These viruses are phylogenetically closely related and belong to the Japanese encephalitis serocomplex group. Similar to other flaviviruses, these viruses are enveloped, with genomes comprising positive-sense, single-stranded RNA approximately 11 kb in length. Upon translation, a single polyprotein is produced, consisting of three structural and seven non-structural proteins. These proteins function in virus binding to the cell membrane, entry into cells, replication, immune evasion, and the production of new virus progeny. Typically, these viruses are maintained in a sylvatic cycle involving avian hosts, such as passerine birds, and mosquitoes. However, they can accidentally spill over to humans through mosquito bites or wildlife exposure. Although humans generally remain asymptomatic and do not support sufficient viral replication for transmission, they can develop febrile disease and, in some cases, severe neuroinvasive diseases, especially among the elderly or immunocompromised individuals. Due to their co-circulation in the same geographical areas and sharing similar hosts and vectors, individuals in Italy and Germany have been detected as seropositive for WNV and USUV, while seropositivity for WNV and SLEV has been observed in the Americas. Viruses in the Japanese encephalitis virus serocomplex group exhibit significant antigenic similarity. The envelope protein alone contains 12 distinct epitopes and at least three highly conserved epitopes among the JEV serocomplex. Consequently, infection with one member of the JEV serocomplex group, such as WNV, induces WNV-specific antibodies and heterotypic antibodies that can cross-neutralize other members of the JEV serocomplex group, such as USUV and SLEV. Therefore, cross-reactive epitopes can protect against heterologous virus challenges to varying extents, depending on the accessibility of the antibodies to the epitopes. Prior infection with WNV or its envelope domain III (EDIII) or non-structural protein 1 (NS1) protected mice from lethal JEV challenges. Vaccination against WNV protected mice from lethal USUV challenges, and vice versa. Immunity to JEV or SLEV protected hamsters from lethal WNV challenges. Although human sera immune to WNV cross-neutralized USUV and SLEV in vitro during serodiagnosis, the actual mechanism of cross-protection among WNV, USUV, and SLEV remains poorly characterized. Therefore, this study aims to understand the mechanism of cross-protection. Specifically, this research investigated whether human plasma immune to WNV could cross-protect mice from encephalitis caused by SLEV or USUV. Initially, WNV-specific human convalescent plasma and mouse WNV convalescent serum (as a positive control) neutralized WNV and cross-neutralized USUV and SLEV in vitro in a neutralization test. Subsequently, immunocompetent mice were intraperitoneally injected with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum the day before being challenged with WNV, SLEV, or USUV via footpad injection. We found that human WNV convalescent plasma provided mice with strong protection against neuroinvasive encephalitis caused by WNV. Additionally, human WNV convalescent plasma reduced the viremia titers of SLEV and USUV for several days during acute infection. Human WNV convalescent serum also demonstrated a trend towards protecting mice from SLEV-induced encephalitis, as evidenced by lower SLEV titers in the brain and histopathology scores. These findings will aid in decoding the mechanisms of cross-protection among the JEV serovars, developing therapeutic strategies against WNV, SLEV, and USUV, and anticipating potential disease outcomes, especially in regions where multiple viruses of the JEV serocomplex are endemic. / Master of Science / West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging flaviviruses transmitted by mosquito bites, primarily among perching birds. However, mosquitoes can also transmit these viruses to animals and humans, especially in regions where these viruses are prevalent. The immune system, which defends against pathogens and other diseases, usually combats these viruses effectively, preventing most people from developing symptoms. The immune system has two main branches: the innate immune system, which confers immediate defense, and the adaptive immune system that includes antibodies and certain long-lasting memory cells, that can fight off infections years after the initial exposure to the same or similar disease-causing agents. Occasionally, the immune system fails to fight these viruses, particularly in the elderly or those with chronic diseases, leading to fever or severe brain inflammation called encephalitis. Currently, WNV and SLEV are circulating in the Americas, while WNV and USUV are present in European countries. Due to similar transmission methods, infection patterns, and geographical overlap, individuals might be sequentially infected with WNV and USUV in Europe, and WNV and SLEV in the Americas in their lifetime. These viruses also share common antigens, which can induce similar immune responses. Therefore, the immune response to one virus might protect against another with similar antigens. It has been reported that the immune response induced by WNV can protect against encephalitis caused by USUV or SLEV. However, it remains unclear whether this cross-protection is mediated by antibodies or a certain type of immune cells called T cells. This study investigates whether antibodies induced by WNV infection can protect against SLEV or USUV in a mouse model. Plasma, the part of blood containing antibodies, is referred to as convalescent plasma when collected after an individual has recovered from an infection or disease. Human WNV convalescent plasma was tested against SLEV and USUV using a plaque reduction neutralization test to determine the antibodies’ ability to prevent viral infection in a laboratory setting. Human WNV convalescent plasma effectively prevented SLEV and USUV from infecting cells. We then developed a mouse model that could be infected with SLEV or USUV and mimic human disease. Groups of mice were systematically transferred with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum one day before the infection with WNV, SLEV, or USUV. Disease conditions, such as weight loss, reduced movement, hunchback, fur loss, and occasional paralysis, were monitored until the infected mice were humanely euthanized. After euthanasia, the brains of the mice were collected to measure viral load and examine signs of encephalitis. We observed asymptomatic disease outcomes reflecting natural human infection. Both human and mouse WNV convalescent samples reduced viral load in the blood for a period in both SLEV and USUV-challenged groups. Mice treated with human WNV convalescent plasma showed a trend of lower SLEV in their brains. Additionally, mice treated with mouse WNV convalescent serum had lower SLEV titers in their brains compared to those treated with mouse normal serum. Overall, these findings suggest that human WNV convalescent plasma provides some crossprotection against SLEV- and USUV-induced diseases. Understanding the mechanism of crossprotection is crucial for developing therapeutics against these viruses and predicting disease outcomes in areas where multiple viruses of the Japanese encephalitis virus serocomplex are prevalent.

Made available in DSpace on 2016-01-26T12:51:48Z (GMT). No. of bitstreams: 1 carolinacolombellipacca_tese.pdf: 2227429 bytes, checksum: 4bcc12b8c06f6322170e32bfccfc8fa1 (MD5) Previous issue date: 2013-11-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introduction. Arboviruses, arthropod-borne viruses, are frequently associated with human outbreaks and represent a serious health problem. The genus Flavivirus, which includes both the Yellow Fever Virus (YFV) and Saint Louis Encephalitis Virus (SLEV), are important pathogens that result in high morbidity and mortality rates worldwide. In Brazil, YFV has a sylvatic cycle and occurs annually, despite the efficiency of the vaccine. Saint Louis Encephalitis is an infectious illness that can cause acute fever caused by SLEV, which is widely distributed in the Americas. The emergence of SLEV became a serious concern after the first related outbreak in Brazil in 2006, in the city of Sao Jose do Rio Preto. There is no specific antiviral drug for these viruses, only supporting treatment that can alleviate the symptoms and prevent complications. The need to develop effective and safe antiviral drugs is indispensable for the treatment of these infections. Objective. The aim of this work was to identify new possible antiviral drugs against the arboviruses that can cause acute fever and encephalitis (YFV and SLEV) and to evaluate the capacity of inhibition of these compounds in ABR mice. Material and Methods. Plaque reduction assay, flow citometry, immunofluorescence and cellular viability were used to test the compounds in vitro. ABR mice were inoculated with YFV, and the biological samples were tested for the presence of the virus through the use of plaque reduction assay and qPCR. Neutralization assay was also performed. Results. Treated cells showed efficient inhibition of viral replication at concentrations that presented minimal toxicity to the cells. The assays showed that ftalyl-tiazole and fenoxytiosemicarbazone were more effective, and that they reduced viral replication by 60% and 75% for YFV and SLEV, respectively. The analysis also revealed that the ABR mice inoculated with YFV had histopathological alterations in the liver; however, the samples did not present viral title. Neutralization assay showed a high concentration of antibodies in the serum. Conclusion. The inhibitions of viral replication were confirmed through the use of some assays in vitro, and the effectiveness of the selected compounds show that they are an option in the treatment of these viruses. More detailed studies are needed to determine the mechanism of action of these molecules. The mice were found to have histopathological alterations, which indicates viral infection; however, they also presented with high concentrations of antibodies. More studies about animal models are necessary to make in vivo experiments. / Introdução: Os arbovírus, vírus transmitidos por artrópodes, são freqüentemente associadas a surtos em seres humanos e representam um problema sério de saúde pública. Os vírus pertencentes ao gênero Flavivirus, tais como vírus da Febre Amarela (YFV) e vírus da Encefalite de Saint Louis (SLEV), são importantes patógenos que podem causar alta taxa de morbidade e mortalidade no mundo. No Brasil, YFV é mantido em ciclo silvestre notificados anualmente, a despeito da segurança e eficiência da vacina. A encefalite de Saint Louis é uma doença infecciosa febril aguda causada pelo SLEV amplamente distribuída nas Américas. A emergência do SLEV passou a ser um fato preocupante no Brasil a partir da constatação do primeiro surto no país em 2006, na cidade de São Jose do Rio Preto. Não existe tratamento específico para estas viroses, somente tratamento de suporte para ajudar a aliviar os sintomas e prevenir complicações. Desta forma, há uma grande necessidade de que sejam desenvolvidos antivirais efetivos e seguros para o tratamento destas infecções. Objetivos: O objetivo deste trabalho foi identificar potenciais compostos antivirais contra os arbovírus causadores de doença febril aguda e encefalites (YFV e SLEV) in vitro e avaliar a capacidade de inibição da replicação viral dos compostos in vivo em camundongos ABR. Materiais e Métodos: Para tanto, foram realizados ensaios de redução de placas, citometria de fluxo, imunofluorescencia, bem como testes de viabilidade celular para as analises in vitro. Além disto, camundongos ABR foram inoculados com YFV e seus materiais biológicos testados para a presença de partículas virais por ensaio de redução de placas e qPCR. Adicionalmente, foi realizado ensaio de neutralização do soro dos animais. Resultados: Celulas tratadas com os compostos mostraram eficiente inibição da replicação viral em concentrações que apresentam baixa citotoxicidade. Os ensaios mostraram que derivados de ftalyl-tiazole e fenoxytiosemicarbazone foram os mais eficazes na ação antiviral, apresentando redução de 60% e 75% para YFV e SLEV, respectivamente. Camundongos ABR inoculados com YFV apresentaram alterações histológicas no fígado, entretanto, não foi constatado título viral nas amostras testadas. O ensaio de neutralização mostra altas concentrações de anticorpos no soro dos animais. Conclusões: A inibição da replicação foi comprovada por vários ensaios in vitro evidenciando as moléculas como potentes alternativas para o tratamento dos vírus. Mais estudos são necessários para a determinação do mecanismo de ação destas moléculas. Os camundongos apresentaram alterações histopatológicas sendo um indicativo de infecção, entretanto, apresentam altas taxas de anticorpos. Mais estudos sobre modelo animal são necessários para a realização de ensaios in vivo.

Thesis (Dr. P.H.)--University of Texas School of Public Health, 2008. Thesis (Dr. P.H.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0973. Adviser: Kristy O. Murray. Includes bibliographical references.

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-09-15T13:37:53Z No. of bitstreams: 1 DISS_2014_Letícia Borges da Silva Heinen.pdf: 4369897 bytes, checksum: 2e00a8b07d093787dbdb2e23dd6ac34e (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-09-19T12:59:17Z (GMT) No. of bitstreams: 1 DISS_2014_Letícia Borges da Silva Heinen.pdf: 4369897 bytes, checksum: 2e00a8b07d093787dbdb2e23dd6ac34e (MD5) / Made available in DSpace on 2017-09-19T12:59:17Z (GMT). No. of bitstreams: 1 DISS_2014_Letícia Borges da Silva Heinen.pdf: 4369897 bytes, checksum: 2e00a8b07d093787dbdb2e23dd6ac34e (MD5) Previous issue date: 2014-03-28 / CNPq / O gênero Flavivirus, família Flaviviridae, alberga arbovírus como os vírus dengue (DENV) e da febre amarela, que possuem importância médica e são envolvidos em epidemias de doença febril em áreas urbanas e rurais em regiões tropicais e subtropicais. No Brasil, atualmente, o DENV e o vírus da encefalite de Saint Louis (SLEV) são os dois flavivírus circulantes em áreas urbanas mais frequentes. Desde a introdução e emergência dos diferentes sorotipos de DENV a partir da década de 1980, extensas epidemias de febre do dengue vêm sendo reladas por todo o país. O SLEV, anteriormente reconhecido apenas em ciclos enzoóticos e com pouca relevância médica no Brasil, tem sido implicado em casos de doença febril durante epidemia de dengue no sudeste do país. O objetivo deste estudo foi investigar a circulação de flavivírus em pacientes com doença febril aguda com suspeita de dengue em Mato Grosso (MT) em 2011 e 2012. Material e Métodos: 604 amostras de soro obtidas entre outubro de 2011 e julho de 2012 de pacientes com doença febril aguda suspeita de dengue com até cinco dias do início dos sintomas em MT foram submetidas à multiplex semi-nested RT-PCR para a pesquisa de flavivírus, como os quatro sorotipos de DENV, SLEV, vírus da febre amarela, do Oeste do Nilo, Rocio, Bussuquara, Iguape e Ilhéus. Amostras positivas foram testadas em pelo menos duas reações independentes de single-nested RT-PCR e submetidas a sequenciamento nucleotídico de região do gene da glicoproteína de envelope para análise filogenética. Resultados: Dentre os 604 pacientes, 315 (52,2 %) foram positivos para DENV-4, 24 (4,0 %) para DENV-1, 3 (0,5 %) para SLEV, 1 (0,2 %) para DENV-2 e 1 (0,2 %) para DENV-3. Todas as amostras eram de pacientes oriundos de áreas urbanas de 17 municípios de MT. Entre as amostras positivas, 9 eram co-infecções entre DENV-1/DENV-4, 1 entre DENV-2/DENV-4, 2 por SLEV/DENV-4 e 1 entre SLEV/DENV-1/DENV-4. Os demais flavivírus pesquisados não foram detectados. Amostras negativas para flavivírus totalizaram 273/604 (45,20 %). Discussão: A ocorrência das arboviroses na população geralmente é subestimada, devido a fatores como quadro clínico inespecífico, infecções inaparentes e ausência de diagnóstico diferencial. O DENV-4 foi introduzido no MT em 2012, responsável pela maior casuística nas cidades da Baixada Cuiabana. Co-infecções são frequentes quando há circulação hiperendêmica dos quatro sorotipos do DENV, situação já relatada no Brasil em Manaus e Rio de Janeiro em 2011. O SLEV foi detectado em pacientes de Cuiabá e Várzea Grande. Infecções por SLEV são primariamente inaparentes ou brandas. Em MT, espécies de Culex e outros vetores deste virus são amplamente dispersas. Como humanos são hospedeiros finais e apresentam baixa viremia, sua ocorrência é provavelmente subestimada. Conclusão: DENV-1 e DENV-4 foram os flavivírus identificados com maior frequência. Os quatro sorotipos do DENV foram detectados em Cuiabá e infecções esporádicas pelo SLEV foram identificadas em pacientes co-infectados com o DENV-4 ou o DENV-4/DENV-1 em Cuiabá e Várzea Grande, indicando que outros arbovírus podem circular silenciosamente durante epidemia de dengue em áreas urbanas em MT. / The genus Flavivirus, Flaviviridae family, comprises arboviruses such as the medical important dengue virus (DENV) and yellow fever virus, involved in febrile illness epidemics in urban and rural areas of tropical and subtropical regions. In Brazil, DENV and Saint Louis encephalitis (SLEV) virus are currently the two most important flaviviruses circulating in urban areas. Since the introduction and emergence of different DENV serotypes in the 1980´s, extensive dengue outbreaks have been reported throughout the country.SLEV, previously recognized only in enzootic cycles without medical relevance in Brazil, has been implicated to febrile illness etiology during dengue fever outbreaks in the Southeast region. The aim of this study was to investigate the circulation of flaviviruses in patients with acute febrile illness suspected of harboring dengue in Mato Grosso (MT) between 2011 and 2012. Material and Methods: 604 serum samples obtained between October 2011 and July 2012 from patients with acute febrile illness suspected of dengue lasting less than 5 days in MT were subjected to multiplex semi-nested RT-PCR for flaviviruses, including all four serotypes of DENV, SLEV Yellow Fever, West Nile, Rocio, Bussuquara, Iguape and Ilheus viruses. Positive samples were tested at least twice in independent single-nested RT-PCR reactions and subjected to nucleotide sequencing of the envelope glycoprotein (E) gene region for phylogenetic analysis. Results: Among 604 patients, 315 (52.2 %) were positive for DENV-4, 24 (4.0 %) for DENV-1, three (0.5 %) for SLEV, one (0.2 %) for DENV-2 and one (0.2 %) for DENV-3. All patients are residents in urban areas of 17 cities of MT. Among then, 9 were co-infections among DENV-1/DENV-4, 1 between DENV-2/DENV-4, two between SLEV/DENV-4 and one with SLEV/DENV-1/DENV-4. The other flaviviruses were not detected. Negative samples for flavivirus totaled 273/604 (45.20 %). Discussion: The occurrence of arboviruses in the population generally is underestimated, probably due to unapparent infection or unspecific clinical presentation, associated to the absence of differential diagnosis. The DENV-4 serotype was introduced in MT in 2012, responsible for the largest number of cases in Cuiabá and Varzea Grande. Co-infections are common when hiperendemic circulation of all four serotypes of DENV is observed. This situation has already been reported in Brazil in Manaus and Rio de Janeiro cities in 2011. Three patients were positive for SLEV in Cuiaba and Várzea Grande. SLEV infections are primarily mild or unapparent. In MT, species of Culex and other vectors are widely dispersed. As humans are final hosts and, therefore, present low titer viremia, the occurrence of SLEV in the population is probably underestimated. Conclusion: DENV-1 and DENV-4 were the most frequently flaviviruses identified. The four DENV serotypes were detected in Cuiaba and sporadic SLEV infections were identified in patients co-infected with DENV-4 or DENV-1/DENV-4 in Cuiaba and Várzea Grande, indicating that other arboviruses may circulate silently during dengue epidemics in urban areas of MT.

Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-04-20T17:14:55Z No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-04-27T17:52:07Z (GMT) No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5) / Made available in DSpace on 2018-04-27T17:52:07Z (GMT). No. of bitstreams: 1 DISS_2015_Otacília Pereira Serra.pdf: 1299760 bytes, checksum: adf22b742c77d45b935dd95c6e46cbda (MD5) Previous issue date: 2015-04-20 / CAPES / FAPEMAT / Arbovírus são transmitidos por artrópodes hematófagos, representando um problema de saúde pública em áreas tropicais. O objetivo deste estudo foi investigar a diversidade de espécies de culicídeos e sua frequência de infecção por Alphavirus e Flavivirus em Cuiabá, MT. Foram realizadas capturas com aspirador de Nasci e puçá entre janeiro e abril de 2013 em três locais de 200 setores censitários, definidos aleatoriamente. Os culicídeos foram identificados com chave dicotômica de Forattini, alocados em pools (1-20 mosquitos) segundo sexo, espécie, data, local de coleta e armazenados a -80˚C. Pools de fêmeas foram submetidos à extração de RNA e DNA total, à multiplex semi-nested-RT-PCR para cinco alphavírus e 11 flavivírus e Nested- PCR para identificação de Culex (Cx.) quinquefasciatus. Amostras positivas para SLEV, DENV-1, -4 e MAYV foram submetidas a single RT-PCR e sequenciamento nucleotídico. Pools positivos para o MAYV foram inoculados em células Vero e submetidos a RT-PCR para o gene de envelope E1 dos alphavirus. Pools positivos para flavivirus foram inoculados em células C6/36 (Flavivirus). Calculou-se a taxa de infecção mínima (MIR). Foram capturados 11.090 mosquitos, 4.556 fêmeas de 14 espécies, perfazendo 610 pools. Foram analisados 171 pools de Aedes (Ae.) aegypti; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp; 6 Limatus sp; 2 Mansonia wilsoni; 5 Psorophora (Ps.) sp; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Encontrou-se 1/171 (MIR=0,92) pool de Ae. aegypti positivo para DENV-1; 1/403 (MIR= 0,2) Cx. quinquefasciatus para SLEV genótipo V-A, 12/403 (MIR=3,5) de Cx. quinquefasciatus, 4/171 (MIR=3,67) de Ae. aegypti para MAYV; destes, cinco pools apresentaram co-infecção com DENV-4. Um produto de 1,3 kb obtido com o protocolo para o gene de envelope de três pools positivos para o MAYV resultou em sequências nucleotidicas inespecíficas. O MAYV foi isolado de dois pools contendo duas fêmeas não ingurgitadas de Ae. aegypti (#958) e duas de Cx. quinquefasciatus (#489). Positividade para DENV-4 foi identificada em 58/171 (MIR=53,35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp, 2/11 (MIR=142,85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor e 1/1 (MIR=1000) Aedes sp. O DENV-4 foi isolado de dois pools contendo três (#329) e 16 (#806) fêmeas de Cx. quinquefasciatus não ingurgitadas. O SLEV, MAYV e os sorotipos do DENV foram identificados em pacientes com suspeita de dengue na cidade de Cuiabá em estudos prévios do Laboratório de Virologia. Experimentalmente, Culex e Aedes spp. são vetores competentes do MAYV. A identificação do vírus em fêmeas não ingurgitadas sugere que estas espécies podem estar envolvidas no ciclo urbano do MAYV em Cuiabá. Dentre os sorotipos do DENV, somente o DENV-1 e o DENV-4 foram identificados em culicídeos. O DENV-4 tem produzido importantes epidemias em MT desde 2012. A positividade para DENV-4 em diferentes espécies de culicídeos pode ser decorrente de infecção natural ou da hematofagia em humanos, pois muitos destes pools apresentavam fêmeas ingurgitadas. Cuiabá possui ecossistema favorável para a ocorrência de arboviroses e proliferação de vetores. Estudos envolvendo vigilância entomológica e virológica são importantes para estimar a situação epidemiológica dos arbovírus no estado. / Arbovirus are transmitted by hematophagous arthropods, posing a public health issue in tropical areas. The aim of this study was to investigate the diversity of culicidae and their frequency of infection by arboviruses from Alphavirus and Flavivirus genus in Cuiabá, MT. To achieve that, culicids were captured with Nasci aspirators and hand net between January and April 2013 in three locations of 200 censitary sectors randomly defined. The specimens were identified using the Forattini dichotomy key, allocated in pools (1-20 mosquitoes), according to sex, species, day and place of capture, and stored at -80˚C. Female pools were subjected to total RNA and DNA extraction and to multiplex semi-nested-RT-PCR for five alphaviruses and 11 flaviviruses and Nested-PCR for Culex (Cx.) quinquefasciatus identification. The pools positive for SLEV, DENV-1, -4 and MAYV were subjected to single RT-PCR and nucleotide sequencing. MAYVpositive pools were inoculated in Vero cells and subjected to RT-PCR for the E1 envelope gene of alphaviruses. Pools positive for flaviviruses were inoculated in C6/36 cells. The minimum infection rate (MIR) was calculated. 11,090 mosquitoes were captured, 4,556 females belonging to 14 species, comprising 610 pools, 171 pools of Aedes (Ae.) aegypti specimens; 1 Ae. albopictus; 1 Aedes sp.; 5 Cx. bidens/interfor; 1 Cx. spinosus; 403 Cx. quinquefasciatus; 1 Galindomyia sp.; 6 Limatus sp.; 2 Mansonia wilsoni; 5 Psorophora sp.; 1 Ps. ciliata; 11 Ps. varipes/albigenu; 1 Sabethes chloropterus e 1 Uranotaenia sp. Among them, 1/171 (MIR=0.92) Ae. aegypti pool was positive for DENV-1 and 1/403 (MIR=0.3) Cx. quinquefasciatus for SLEV genotype V-A. For MAYV, 12/403 (MIR=3.5) Cx. quinquefasciatus, 4/171 (MIR=3.67) Ae. aegypti were positive, five of them were also infected by DENV-4. A DNA product with 1.3 kb obtained from three pools positive for MAYV with the protocol for the envelope gene resulted in unspecific nucleotide sequences. MAYV was isolated from two pools, both containing two non-engorged females of Ae. aegypti (#958) and Cx. quinquefasciatus (#489). DENV-4 was detected in 58/171 (MIR=53.35) Ae. aegytpi, 105/403 (MIR=30,65) Cx. quinquefasciatus, 2/5 (MIR=400) Psorophora sp., 2/11 (MIR=142.85) Ps. varipes/albigenu, 1/1 (MIR= 1000) Sabethes chloropterus, 2/5 (MIR=285.7) Cx. bidens/interfor and 1/1 (MIR=1000) Aedes sp. DENV-4 was isolated from two pools containing three (#329) and 16 (#806) non-engorged females of Cx. quinquefasciatus. The SLEV, MAYV and the four DENV serotypes were identified in patients suspected of harboring dengue infection in Cuiabá in previous studies of the Virology Laboratory. Experimentally, Culex and Aedes spp. are competent vectors for MAYV. The identification of the virus in nonengorged females suggests these species may be involved in the urban cycle of MAYV in Cuiabá. Among the DENV serotypes, only DENV-1 and DENV-4 were identified in culicids captured in the city. DENV-4 has been responsible for major outbreaks in MT since 2012. The identification of DENV-4 in several mosquito species might be resultant either from natural infection or hematophagy in humans, since several of these pools presented engorged females. Cuiabá presents a favorable ecosystem for the occurrence of arboviruses and vector proliferation. Studies involving entomological and virological surveillance are important to estimate the epidemiological situation of arboviruses in the state.

Made available in DSpace on 2016-01-26T12:51:28Z (GMT). No. of bitstreams: 1 adrianomondini_tese.pdf: 12162182 bytes, checksum: fe0a4d238020f614624084ebb47e1011 (MD5) Previous issue date: 2010-03-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dengue belongs to the Flavivirus genus and is the most common arboviral infection worldwide. It can be caused by four antigenically different serotypes (DENV 1-4). These serotypes are transmitted mainly by the bite of Aedes aegypti mosquitoes. The vector is widely associated with human activity and the influence of organized social space favors the interaction among vector, virus and man, making populated areas sources of dengue dispersion. In this study, we performed a molecular, spatial and temporal study of DENV transmission through positive samples of blood and infected mosquitoes captured in São José do Rio Preto/SP in a period of four years. Material and Methods: Serum samples of patients presenting dengue like symptoms and pools of mosquitoes had their viral RNA extracted and were tested by Multiplex- RT-PCR with Flavivirus generic primers based on non-structural protein (NS5) in the first round, followed by Nested assays with species-specific primers for the identification of DENV 1-3, yellow fever virus, Saint Louis encephalitis virus (SLEV) among others. Positive samples were analyzed spatially and phylogenetically. Results and Discussion: We analyzed 613 blood samples for four years: 199 in 2006, 94 in 2007, 313 in 2008 and 10 in 2009. The positivity was high in 2006 and 2007, with 106 and 51 infected patients, respectively. The major dengue serotype circulating during the 2006 and 2007 epidemics was DENV-3 and few cases of DENV-2, which is an indication of its recent introduction in the municipality. We also reported the first outbreak of SLEV in Brazil in 2006. Among DENV patients in 2008, only seven were infected by DENV-3 and 90 were infected by DENV-2, suggesting the reemergence of this serotype. We detected the circulation of DENV-1 in two Abstract xxv patients in 2008 and in four patients in 2009. Nearly 1200 mosquitoes were captured from December 2007 to March 2008. We have captured 814 Aedes aegypti mosquitoes, which were divided in 463 pools. Only 3.67% of them were positive for DENV-3 and DENV-2. Pools containing only male mosquitoes were positive for DENV, indicating the presence of transovarial transmission. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000 2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area. / Dengue pertence ao gênero Flavivirus e é a infecção por arbovírus mais comum no mundo todo. Pode ser causada por quatro sorotipos antigenicamente distintos (DENV 1-4). Estes sorotipos são transmitidos pela picada do mosquito Aedes aegypti. O vetor está amplamente associado a atividade humana e a influencia do espaço urbano favorece a interação entre o vetor, o vírus e o homem, tornando áreas populosas, grandes centros de dispersão do dengue. Neste estudo, foi realizada um estudo molecular, espacial e temporal da transmissão de DENV através de amostras positivas de sangue e de mosquitos infectados capturados em São José do Rio Preto/SP, num período de quatro anos. Materiais e métodos: Soro de pacientes apresentando sintomas de dengue e pools de mosquitos tiveram seu RNA viral extraído e foram testados por Multiplex-RT-PCR, com primers genéricos de Flavivirus baseados na proteína não estrutural 5 (NS5) numa primeiro ciclo,seguida por ensaios Nested com primers específicos para DENV, para o vírus da febre amarela, para o vírus da encefalite de Saint Louis, entre outros. As amostras positivas foram analisadas espacial e filogeneticamente. Resultados e discussão: Analisamos 613 amostras de soro durante 4 anos: 199 em 2006; 94 em 2007; 313 em 2008 e 10 em 2009. A positividade foi alta em 2006 e 2007, com 106 e 51 pacientes infectados, respectivamente. O principal sorotipo circulante durante as epidemias de 2006-2007 foi DENV-3 e poucos casos de DENV-2, o que pode ser a indicação de sua recente introdução no município. Nós também descrevemos a primeira epidemia de SLEV no Brasil em 2006. Dentre os pacientes com DENV em 2008, apenas sete estavam infectados com DENV-3 e 90 com DENV-2, sugerindo a reemergência do sorotipo. Nós Resumo xxiii detectamos a circulação de DENV-1 em dois pacientes em 2009 e em quatro pacientes em 2009. Aproximadamente 1200 mosquitos foram capturados entre Dezembro 2007 e Março de 2008. Capturamos 814 mosquitos Aedes aegypti, que foram divididos em 463 pools. Apenas 3,67% deles foram positivos para DENV-2 e DENV-3. Pools contendo apenas machos foram positivos para DENV, indicando a presença de transmissão transovariana. Nós obtivemos sequências de 82 pacientes dentre 174 amostras de sangue. Nós fomos capazes de geocodificar 46 sequências. O alinhamento gerou gerou nucleotídeos com 399 bp com 134 taxa. A análise filogenética indicou que todas as amostras foram de DENV-3 e estavam relacionadas às cepas circulantes na ilha da Martinica em 2000-2001. Sessenta pacientes com DENV- 3 de São José do Rio Preto formaram um grupo monofilético (linhagem 1), intimamente relacionado com os outros 22 isolados (linhagem 2). Nós assumimos que estas linhagens apareceram antes de 2006 em ocasiões diferentes. A possibilidade de inferir a dinâmica espaço-temporal através de dados genéticos é relativamente pouco explorada e pode esclarecer acirculação de DENV. O uso de dados filogenéticos estruturadosgeograficamente e temporalmente forneceu uma visão detalhada na dispersão de, pelo menos, duas cepas virais distintas numa área urbana.